College of Veterinary Sciences LahoreGroup members names:

Presentation

Tissue Preparation

Dr.Nabeel Yousaf

Tissue PreparationIntroduction “Tissue processing” describes the steps required to take animal

or human tissue from fixation to the state where it is completely infiltrated with a suitable histological wax and can be embedded ready for section cutting on the microtome.

Importance Microscopic analysis of cells and tissues requires the

preparation of very thin, high quality sections (slices) mounted on glass slides and appropriately stained to demonstrate normal and abnormal structures.

• It involves several steps that are following.1. Obtaining a fresh specimen 4. Clearing2. Fixation 5. Wax infiltration3. Dehydration  6. Embedding or

blocking out

1. Obtaining a fresh specimen

• Fresh tissue specimens will come from various sources.

• A sharp blade or knife must be used for tissue collection.

• Obtain a thin layer of tissues about 1 cm thick.

• Care full handling • Fix the specimen as soon possible.• Wash specimen with normal saline for

maximum penetration of fixative.

2. FixationTo maintain the natural state of specimen.Fixation facilitates proper staining of tissue.Fixative should be 20-25 times of volume of specimen.Characters of a good fixative:* Penetrate quickly * Prevent tissue from shrinkage• Good fixatives:• Formaldehyde • Alcohol• Picric Acid• Potassium Dichromate etc.

WashingFor removal of fixative.6-8 hours.Under tap water.

3. Dehydration|Removal of water. Alcohols are most commonly used for

dehydration.Water replacement with alcohol.Different % ages of alcohol are used for

different time periods.30%,50%,70% Alcohol for 2,2 hours.80%,95% for 1,1 hours.95% for 2 hours.100% for ½ hours two times.

4. ClearingRemoval of dehydrating agent.Clearing agent > miscible with both embedding medium and dehydrating agent.Zylene ( clearing agent )Tow times for 1,1 hours.Other reagents :Toluene, Chloroform, Benzene, Cedar wood oil etc.

5. Wax infiltration

Replace xylene with paraffin.Immerse in melted paraffin.Remove all bubbles, xylene. Procedure Two baths of melted paraffin. (a) Leave the tissue for 1 hour in malted paraffin. (b) Put in fresh paraffin and melt it again.

6. Embedding or blocking out

Allow the melted wax to be solidify.It get rigid.A. Orient tissue 1. cross section 2. longitudinal section

B. Dissection orientationC. Avoid bubbles

EmbeddingProcedure 1. Place tissue cassette in melted paraffin 2. Fill mold with paraffin 3. Place tissue in mold 4. Allow to cool

Sectioning – Trimming the BlockUntrimmed tissue block

Trimmed block with excess paraffin removed and block face in a trapezoid shape

SectioningProcedure 1. Place tissue block in microtome with wide

edge of trapezoid lowest, and parallel to knife 2. Advance blade toward block 3. Begin sectioning

Mounting sectionsA. 40o C water bath 1. Flattens paraffin section 2. Permits mounting on slideB. Gelatin & albuminC. Glass slidesD. Oven / air dry

StainingProcedure1. Slide rack 2. Solutions a. rehydration b. stain c. dehydration

CoverslippingA. Coverslip & mounting medium (not miscible

with water)

B. DehydrateC. Clearing agentD. Permount

Pitfalls. Poor fixation (poor structural details)

B. Inadequate dehydration

C. Contaminated xylene (milky)

D. Poor infiltration (bubbles, poor support)

E. Embedding: orientation, bubbles

Pitfalls. Coverslipping 1. Bubbles

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